0 software. Immunostaining The nuclei of maize roots were prepared in the slides ac cording to the reported method. Immunostaining of the nuclei on the slides was carried out as described by Tofacitinib mw Zhang et al. The primary antibodies were H3K9Ac and H4K5Ac and the secondary antibody was fluores cein conjugated goat anti rabbit IgG. In control exper iments, slides were incubated with the secondary antibody only. All slides were counterstained with 4,6 diamidino 2 phenylindole, mounted with Vectashield. Images were captured with a CCD monochrome camera Sensys 1401E under an Olympus BX 60 fluorescence micro scope with filter blocks for DAPI and fluorescein, then pseudo Inhibitors,Modulators,Libraries colored and merged using the software Meta Morph 7. 7. 2.
Micro scope settings and camera detector exposure times were kept constant for the control and treated groups and more than 300 nuclei were analyzed. Images were proc essed using ADOBE PHOTOSHOP 9. 0 software. The mean Inhibitors,Modulators,Libraries gray value of the im munostaining signals for H3K9Ac and H4K5Ac in the control and NaCl treated samples was measured with Image J and MetaMorph. For both the control and treated groups, three independent immunostaining ex periments were performed with each antibody. Mean gray value of the signal intensity and standard error of the mean value were calculated with SPSS10. 0 for Win dows package. Western blot assay Proteins were extracted from maize seedling roots by grinding in the liquid nitrogen and resuspended in the extraction buffer. Western blot detection was carried out as described by Yang et al.
Proteins were fractionated by SDS PAGE and transferred to Immobilon P membranes which were respectively incubated with the primary anti bodies H3, H3K9Ac and H4K5Ac overnight at 4 C. Detection was performed using alkaline phosphat ase conjugated anti rabbit IgG antibody and chemiluminescence Inhibitors,Modulators,Libraries visualization. Histone H3 was ap plied as an equal loading control. Densitometric mea surements were taken after immunodetection using Image J. Abundance index was calculated as follows H3K9Ac or H4K5Ac band intensity/H3 band intensity. Western blots were repeated three times for each sample from three independent experiments. Mean abundance index and standard error of the mean were calculated with SPSS10. 0 for Windows package. Quantitative real time PCR Total RNA was isolated with Trizol reagent.
The purified RNA was reverse transcripted Inhibitors,Modulators,Libraries to cDNA by using Inhibitors,Modulators,Libraries RevertAid First Strand cDNA Synthesis sellekchem Kit. The reverse transcription product was diluted by ten times to perform real time PCR amplification reaction in triplicate for tech nical repeats. Quantitative real time polymerase chain reaction was carried out using SYBR Green Real time PCR Master Mix in an ABI StepOne Plus real time PCR system with the following cycling conditions 94 C for 1 min, followed by 40 amplification cycles at 94 C for 15 s, 56 C for 30 s and 72 C for 30 s.